We have shown that the HD of IBV E plays an important role in the release of infectious IBV particles from Vero cells. A recombinant virus with IBV E containing a heterologous HD (EG3) was competent for virus assembly but showed a defect in the release of infectious particles. The finding that the HD was not required for assembly is consistent with our earlier observation that the cytoplasmic tail of IBV E is sufficient for interaction with IBV M (4
). Further characterization of the mutant virus showed that it accumulated intracellularly in vacuole-like structures along with aberrant material. We hypothesized that the mutant virions were accumulating intracellularly and becoming damaged and were subsequently released as noninfectious particles. Thus, we initially thought that the HD of IBV E might alter the secretory pathway to promote anterograde trafficking. However, in overexpression experiments IBV E, but not EG3, caused a dramatic reduction in protein trafficking to the plasma membrane by impeding cargo trafficking through the Golgi complex. We also observed that overexpression of IBV E disrupted Golgi morphology but did not affect the ER or endosomal compartments. Finally, we observed that cells infected with IBV-EG3 had increased surface levels of IBV S, leading to larger syncytia.
Previously our lab reported that expression of IBV E using a recombinant vaccinia virus in BHK-21 cells did not disrupt Golgi structure (5
). In the current study we expressed IBV E from a plasmid using lipid-based transient transfection of HeLa cells. One possible reason for the difference we observed in the previous study could be the different cell types used. We tested this by overexpressing IBV E via lipid-based plasmid transfection in several different cell lines, including BKH-21. In all cases, IBV E disrupted the Golgi morphology (data not shown). We next tested whether the lipid-based transfection method was responsible for the effects that we observed. We used a CaPO4
-based transfection method as well as nucleofection and observed the same effects on the Golgi complex as we saw using lipid-based methods (data not shown). Thus, the discrepancy in the data is likely due to the method used to express IBV E. Vaccinia virus expression produces a large amount of protein very rapidly, whereas transient transfection tends to produce a more modest amount of protein over a longer period of time.
Our lab previously reported that IBV does not replicate efficiently when IBV S accumulates on the surface of cells early in infection (39
). The ability of IBV E to reduce protein trafficking is likely beneficial to the virus because it prevents the accumulation of IBV S on the surface of infected cells, thereby reducing syncytium size and number. Large syncytia may die prematurely, which prevents robust virus replication. Increased syncytium size may also make virion trafficking more challenging due to the intracellular rearrangements caused by cell-cell fusion. Furthermore, reducing protein trafficking during host infection may have other positive effects for the virus, such as reducing the amount of antigen on the cell surface or preventing antigen display to the immune system by the major histocompatibility complex I.
The importance of the E protein in the release of infectious particles has been observed for other CoVs. Mutations introduced into the HD of MHV E via alanine scanning produced mutant viruses which, among other defects, were compromised in release of infectious virus (37
). Studies investigating the role of transmissible gastroenteritis coronavirus (TGEV) E showed that when E protein was deleted from the virus, virions accumulated intracellularly, and infectious virus could not be recovered unless E was provided in trans
). These results, combined with our data showing that IBV E alters the secretory pathway, suggest that the CoV E protein supports the release of infectious particles.
The apparent disassembly of the Golgi complex in response to expression of IBV E raises some interesting questions. Previously it has been reported that MHV infection drives the rearrangement of the Golgi complex in a two-step process, where initially the Golgi complex is dispersed from its juxtanuclear position by an unknown mechanism, followed by the condensation of the Golgi complex in the centers of syncytia, seemingly driven by cell-cell fusion (10
). More recent work by Ulasli et al. has greatly expanded our understanding of the membrane rearrangements caused by coronaviruses. These authors describe the formation of large virion-containing vacuoles from ERGIC/Golgi membranes concurrent with the scattering of the Golgi complex (30
). These results suggest that the rearrangement of the Golgi complex may be important for forming virion carriers. It is interesting to speculate on why the virus would need to alter the secretory pathway in order to properly traffic its virions. Virions are much larger than normal cargo and may traverse the secretory pathway using a different route, or they may require different machinery than conventional cargo. The morphological changes in the Golgi complex that we observed in the presence of IBV E may help to create an environment that promotes virion trafficking.
The mechanism by which IBV E modifies the secretory pathway is not understood. One possibility is that IBV E oligomerizes and forms an ion channel at the Golgi complex. Other viruses encode small hydrophobic proteins that oligomerize and form ion channels, with the best characterized being influenza virus M2 (23
). The proton channel activity of influenza virus M2 plays an important role during the entry of influenza virus by acidifying the lumen of the virion following endocytosis, which allows for uncoating of the virus (34
). For some strains of influenza virus, M2 also acts at the trans
-Golgi network (TGN), where it raises the pH of the TGN lumen to prevent the premature activation of the fusion protein, hemagglutinin (27
). A consequence of increasing the TGN pH is that normal protein trafficking is slowed both through the Golgi complex and to the plasma membrane (25
). It is possible that IBV E follows a similar paradigm for ion channel activity in the secretory pathway. In vitro
data suggest that an IBV E channel would likely conduct Na+
). If IBV E disrupts the Na+
gradient at the Golgi complex, it could have a deleterious impact on the Na+
exchangers at the Golgi complex that are critical for maintaining the proper lumenal pH of the Golgi complex (19
). It is also conceivable that the presence of an IBV E ion channel at the Golgi complex could affect the homeostasis of other ions critical to Golgi function, such as Ca2+
). In addition to the effect on protein trafficking, changing the ion balance within the Golgi complex could inactivate proteases that damage virions. This would explain why IBV S is damaged in cells infected with IBV-EG3 and not in those infected with IBV-wt. Additionally, a change in the lumenal ion concentration could have an effect on the structure of the Golgi complex. It is clear that more work will be required to understand the putative ion channel activity of IBV E. This information will be essential for understanding the function of IBV E at the Golgi complex.
While there are considerable in vitro
data focused on the ion channel activity of CoV E, it has not yet been demonstrated that the CoV E protein possesses ion channel activity in infected cells. Thus, it is important to consider other possibilities for the mechanism of IBV E function. It may be that the HD of IBV E facilitates a protein-protein interaction that affects protein trafficking. One possibility is that the HD of IBV E interacts with the membrane domain of SNARES that regulate vesicle fusion at the Golgi complex. It is also possible that IBV E may interact with cellular ion channels present in Golgi membranes and modulate their activity. For example, if the HD of IBV E was able to bind tightly to the V0
subunit of the V-ATPase, it could prevent assembly of the active pump and render it unable to acidify the Golgi lumen. This would lead to trafficking and morphological changes similar to those caused by overexpression of IBV E (38
). Finally, by mutating the HD of IBV E, we may have disrupted the formation of IBV E oligomers. While oligomerization would be required for ion channel activity, it may also be important for the types of protein-protein interactions described above.
While the CoV E proteins are small, they appear to have multiple functions. Previous characterization of IBV E showed that the C-terminal tail of the protein contains targeting information and facilitates interaction with IBV M (3
). Here, we took advantage of a mutant version of IBV E that was competent for assembly but defective in release of infectious particles. We showed that the HD of IBV E alters the cellular secretory pathway. This indicates that multiple domains of IBV E are important for its proper function, and this is possibly true for all CoV E proteins. Future studies will examine which residues within the HD of IBV E are critical for its effect on the secretory pathway. Additionally, it will be important to determine how IBV alters the secretory pathway through direct ion channel activity, protein-protein interaction, or some other mechanism.